PROJECT SUMMARY This proposal focuses on mononuclear nonheme iron complexes and enzymes that activate dioxygen and oxygenate substrates. An important class of nonheme iron enzymes is the thiol dioxygenases, which utilize a single iron center and O2 to oxidize thiol substrates to sulfinic acids. Mammalian cysteine dioxygenase (CDO) and bacterial 3-mercaptopropionate dioxygenase (p3MDO) are two enzymes in this class. Proper functioning of CDO is important for maintaining the appropriate levels of cysteine and producing cysteine sulfinic acid as part of cysteine metabolism in mammals. Loss of CDO function has been linked to a number of diseases including Parkinson's and Alzheimer's disease, as well as certain types of cancer. The mechanism of action of these enzymes is poorly understood. Efforts described in this proposal include the design and synthesis of a new series of iron complexes that activate O2 and carry out selective substrate oxidation reactions including S-oxygenation, similar to CDO and p3MDO. This work is also relevant to the larger class of nonheme iron oxygenases. The modular organic ligand scaffold surrounding the metal ion will be rationally adjusted to examine structure/function relationships. The study of these complexes will provide fundamental knowledge that will contribute to delineating enzyme mechanisms and to designing selective biomimetic iron oxidation catalysts. The O2 reactivity of new nonheme iron complexes bearing sulfur ligands will be examined by methods designed to trap and/or characterize unstable Fe/O2-derived species. These methods include the use of low temperatures to trap unstable species during O2 activation, and spectroscopic methods such as stopped-flow UV-vis coupled with rapid-freeze-quench trapping, resonance Raman, EPR, low-temperature ESIMS, and Mssbauer. Density functional theory (DFT) calculations will support and guide the spectroscopic and mechanistic studies. A key aspect of the proposed work also includes select, parallel studies on the enzymes CDO and p3MDO. New oxygen intermediates will be targeted, including the characterization of a promising O2-derived transient species already observed for CDO. The nitric oxide chemistry (NO) of both synthetic complexes and enzymes will be studied, as NO is a well-known and informative surrogate for O2. The spin states and electronic structures of the new FeNO species will be determined, and second and third sphere interactions in CDO will be assessed. The fundamental knowledge to be obtained should provide major advances in our understanding of the mechanisms of a large class of nonheme iron oxygenases/oxidases.